Nucleic acid sequence based amplification pdf

Step 2: The primers anneal to the single-stranded DNA. Step 3: The primers extended by DNA polymerase; one duplex is amplified and converted to two duplexes. The double-stranded DNAs are separated by helicase and this chain reaction repeats itself. RAM is a novel isothermal nucleic acid amplification method. This technique is termed as RAM because the amplification power is derived from primer extension, strand displacement and multiple ramification branching points.

The multimeric ssDNA generated then serves as a template where multiple reverse primers hybridize, extend and displace downstream DNA and generate a large ramified branching DNA complex.

This ramification process continues until all ssDNAs become double-stranded, resulting in an exponential amplification that distinguishes itself from the previously described nonexponential RCA [ Figure 8 ]. It can be readily used in clinical laboratories for the detection of genes and infectious agents in various areas, such as Hematology, Oncology, infectious disease, Pathology, forensics, blood banks and genetic disease.

Each alternative method has their unique distinguishable properties as well as they have some common property.

Each of the alternate isothermal methods has their own limitations and their mechanisms are relatively complex, they are easy to perform and offer better sensitivity. Incorporation of real-time detection methods such as ECL, molecular beacon made them more competent to be used widely. In the future, some of these methods will be as acceptable and applicable as PCR. Source of Support: Nil. Conflict of Interest: None declared. National Center for Biotechnology Information , U. Search database Search term.

J Pharm Bioallied Sci. Nur Hossain , Sumaiya Islam , and Md. Alimuddin Chowdhury 3. Nur Hossain. Alimuddin Chowdhury. Author information Article notes Copyright and License information Disclaimer. Address for correspondence: Mr. Fakruddin E-mail: moc. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.

This article has been cited by other articles in PMC. Abstract Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. KEY WORDS: Amplification methods, ligase chain reaction, loop mediated isothermal amplification, multiple displacement amplification, nucleic acid sequence based amplification, polymerase chain reaction alternatives.

Loop Mediated Isothermal Amplification LAMP is a specific, simple, rapid and cost-effective isothermal nucleic acid amplification method. Open in a separate window. Figure 1. Schematic description of loop mediated isothermal amplification assay. Figure 2. Strand Displacement Amplification SDA, first described in ,[ 6 ] is an isothermic amplification method, which utilizes four different primers of which a primer containing a restriction site a recognition sequence for HincII exonuclease is annealed to the DNA template.

Figure 3. Target generation scheme for strand displacement amplification. Figure 4. Schematic representation of multiple displacement amplification mechanism. Figure 5. Figure 6. Helicase dependant amplification HDA is an isothermal nucleic acid amplification method using the replication fork mechanism. Figure 7. Ramification amplification method RAM is a novel isothermal nucleic acid amplification method. Figure 8.

Schematic representation of ramification amplification of ligated circular probe. Comparison of nucleic acid amplification methods Each alternative method has their unique distinguishable properties as well as they have some common property. Table 1 Comparison of nucleic acid amplification methods. Conclusion Each of the alternate isothermal methods has their own limitations and their mechanisms are relatively complex, they are easy to perform and offer better sensitivity.

References 1. Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am. Fakruddin M. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Compton J. Nucleic acid sequence-based amplification.

Walker GT. Empirical aspects of strand displacement amplification. PCR Methods Appl. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet. Ligase chain reaction LCR - overview and applications.

Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. Fakruddin M, Chowdhury A. Pyrosequencing: An alternative to traditional sanger sequencing. Am J Bio Biotechnol. Pyrosequencing-prospects and applications.

Gill P, Ghaemi A. Nucleic acid isothermal amplification technologies: A review. Nucleosides Nucleotides Nucleic Acids. Sooknanan R, Malek LT. Challenges to developing nucleic acid sequence based amplification technology for the detection of microbial pathogens in food.

Rev Med Vet. Manojkumar R, Mrudula V. Applications of real-time reverse transcription polymerase chain reaction in clinical virology laboratories for the diagnosis of human diseases.

Am J Infect Dis. Strand displacement amplification - An isothermal, in vitro DNA amplification technique.

Prospective evaluation of BDProbeTec strand displacement amplification SDA system for diagnosis of tuberculosis in non-respiratory and respiratory samples. J Med Microbiol. Real-time, sequence-specific detection of nucleic acids during strand displacement amplification. Anal Biochem. Whole genome amplification - Applications and advances. Curr Opin Biotechnol. The use of whole genome amplification in the study of human disease. Prog Biophys Mol Biol. Genome Res. Eur Food Res Technol.

Lasken RS, Egholm M. Whole genome amplification: Abundant supplies of DNA from precious samples or clinical specimens. Trends Biotechnol. Comprehensive human genome amplification using multiple displacement amplification. Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection. Detection of multiple allergen-specific IgEs on microarrays by immunoassay with rolling circle amplification. Clin Chem. Schweitzer B, Kingsmore S.

Combining nucleic acid amplification and detection. Using a deoxyribozyme ligase and rolling circle amplification to detect a non-nucleic acid analyte, ATP. J Am Chem Soc. Demidov VV. Rolling-circle amplification in DNA diagnostics: The power of simplicity. Expert Rev Mol Diagn. Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification. The mechanism and improvements to the isothermal amplification of nucleic acids, at a glance.

Analytical biochemistry. Nucleic acid-based biotechnologies for food-borne pathogen detection using routine time-intensive culture-based methods and fast molecular diagnostics. Food Science and Biotechnology. Molecular techniques of viral diagnosis.

Viral infections are the cause of very serious problems in humans all over the globe. View 2 excerpts, cites methods. Amplification chemistries in clinical virology.

Computer Science, Medicine. Journal of Clinical Virology. Immuno- and nucleic acid-based current technique for Salmonella detection in food. European Food Research and Technology. Recent advances in molecular techniques for the identification of phytopathogenic fungi — a mini review. Highly Influenced. View 3 excerpts, cites background. Protein detection through different platforms of immuno-loop-mediated isothermal amplification. Materials Science, Medicine.

Nanoscale Research Letters. ACS sensors. View 1 excerpt, references methods. Molecular biotechnology. Highly Influential. View 6 excerpts, references background and methods. View 1 excerpt, references background. Journal of Clinical Microbiology. View 3 excerpts, references background.